Background & objectives: Japanese encephalitis (JE) is a mosquito-borne zoonotic disease. The JE virus (JEV) does not cause any disease among its natural hosts and transmission continues through mosquitoes belonging to Culex vishnui subgroup. This study was aimed to investigate the prevalence of JEV in mosquitoes and humans in the Thanjavur district, a non-endemic region for JE, in Tamil Nadu, by using standard available assays.Methods : A sero-surveillance study was conducted in Thanjavur district among the normal rural school children in the 5–12 yr age group, during the JE season (October) and post-JE season (February) from 2011 to 2013 for the detection of JEV infection. Vector abundance studies were carried out from 2011 to 2014. JE seropositivity and its association between the seasons were analysed statistically.Results : The occurrence of JE infection among children aged 5–12 yr was very high in the study area. The infection rates for JE in two consecutive seasons for 2011–12 and 2012–13 were 32.2 and 65.2%, respectively. The Cx. tritaeniorhynchus sp. dominated the catch, and was majorly responsible for the transmission. There was a significant difference in the human infection rate compared to the years 1991–92 and 1992–93; and a marked decrease in the cattle to pigs ratio (123 : 1) compared to the studies in 1991–93.Interpretation & conclusion : The study unearthed the prevailing situation of JE among children, who are at higher risk of developing the disease during the transmission season. The decrease in the cattle to pigs ratio might be the one of the reasons for increase in the JEV infection among the children population compared to 20 years before. This trend requires urgent attention as it could be prevented with effective surveillance systems and vaccines.

Japanese encephalitis virus (JEV), the causative agent of viral encephalitis is a major public health threat in most parts of East and Southeast Asia[1]. Epidemics of Japanese encephalitis (JE) in many parts of India have now become an annual feature and continue to spread to new areas. In Tamil Nadu, JE is endemic in most of the northern districts[2]. JE cases have been reported during 2007–09 from Chennai, Tiruvallur, Kancheepuram, Vellore and Tiruvannamali districts soon after monsoon months during August and September[3]. The Culex vishnui subgroup of mosquitoes consisting of Cx. triaeniorhynchus, Cx. vishnui and Cx. pseudovishnui have been implicated as major vectors of JE. This group of mosquitoes is extremely widespread and mainly breeds in paddy fields, and the abundance is associated with rice cultivation. Though, Thanjavur district in Tamil Nadu, an important agricultural centre located in the Cauvery delta and known as the “Rice bowl of Tamil Nadu” has not witnessed any JE epidemic and does not support JEV transmission between mosquitoes, reservoirs and amplifying hosts. To monitor JE virus activities in endemic areas, a JE surveillance network programme was started in 1995 in nine districts of Tamil Nadu which included eight endemic zones and one non-endemic zone. The results of infection of the positive pools were intimated to the concerned health authorities to take necessary control measures. After the implementation of this network programme in 1999, there was a considerable decline in the number of human JE cases in Tamil Nadu. However, since 2003 an interesting phenomenon has been observed by the Centre for Research in Medical Entomology (CRME), Madurai in non-endemic zone of Thanjavur district; in which the JEV infection was recorded in desiccated mosquito pools (Minimum infection rate, MIR=1.3) but, it was not recorded in humans. There was no significant difference in overall infection rate (MIR) between non-endemic and endemic areas, between 2000 and 2004 indicating occurrence of silent JEV transmission in Thanjavur[4]. Although high vector abundance and JE virus infection in vector continued to persist, no clinical cases were reported from the area. In view of these observations, it is necessary to investigate this unique phenomenon of silent JE transmission in Thanjavur district to avoid any impending JE outbreak in this non-endemic area of Tamil Nadu. Hence, this study was carried out to find out the factors responsible for the prevailing conditions of JE in Thanjavur district, Tamil Nadu.

Material & Methods

Study area

The present study was conducted in Thanjavur district of Tamil Nadu which is an important centre of South Indian culture, art and architecture. Thanjavur experiences a tropical climate characterized by moderate summer and mild winter with little temperature variations in different seasons. The summer season begins from March and lasts till the middle of June; temperature goes up to 40°C. Thanjavur district receives rain showers from June to September under the influence of the southwest monsoon, and heavier rainfall from October to December from the northeast monsoon. The average rainfall is about 111.37 mm. Rainfall data of Thanjavur district ranged from 686 mm to 971 mm during 2011–13. The district is an important agricultural centre located in the Cauvery delta and the area under rice cultivation ranged from 1.4 to 1.9 lakh ha for the past 10 years[5]. Animal census[6] indicated the ratio of cattle to pigs as 123 : 1.

Sample collection and study design

A sero-surveillance study was conducted among the normal rural school children during JE season (October) and post-JE season (February) from 2011 to 2013 (five times) for the detection of JEV infection, mainly in the 5–12 yr age group studying in seven schools of six villages (Panchayat Union Middle School, Kovilur; Adi Dravida Welfare Primary School, Kovilur; Panchayat Union Middle School, Neduvakottai; Panchayat Union Middle School, Kakkaraikottai; Primary School, Oratharayankudikadu; TAAD Middle School, Thiruvidaimaruthur; and Kalaimagal Primary School, Muthur) in Thanjavur district [Figure 1].

Blood samples collected (after obtaining necessary consents) from each child were used to detect JEV infection. Children in selected villages were followed as a cohort consecutively during JE and post JE seasons from 2011 to 2013. This study was designed based on similar earlier studies already conducted and samples were tested with hemagglutination inhibition (HI) test as previously described[7],[8],[9]. The matched pair test was conducted among children who provided blood samples in both the seasons.

Ethical statement

The study was approved by the 10th Institutional Ethical Committee meeting of CRME, Madurai held on 1 February 2011 (S. No. 3). Written consents of the parents and the respective school teachers were obtained before blood collection and the children were prospectively followed-up.

Vector studies

A longitudinal study of vector abundance was conducted from 2011–14 in three villages of Thanjavur district, namely Muthur, Keezhapattu and Kovilur. During the vector surveillance, two collectors moved freely throughout the village to collect both blood engorged and unfed adult female culicines resting on bushes and thatched roofs of cattle sheds and human dwellings. There were no fixed sites for mosquito collections. Mosquitoes were collected for 1 h after dusk by aspiration and flashlight, transported to the field laboratory, anesthetized with ether, identified to species level and sorted on ice into pools of maximum 50 specimens each for screening. Unfed mosquitoes were pooled, whereas engorged females were held for 48 h for digestion of blood meals before pooling. A key prepared by Reuben et al[10] was used for identification of members of the Cx. vishnui subgroup. Voucher specimens were deposited in the CRME, Madurai museum and the mosquito pools were screened for the virus infection[11],[12],[13],[14].

Statistical analysis

The matched pair test (matched odds ratio and 95% confidence interval) was used to find out association between seasons of same years (JE season–October and post-JE season–February) and between years in the JE seropositivity in the school children. MacNemar test was used to find out the variation in JE seropositivity between the seasons. The analysis was done by EpiInfo software ver. 7.0 (CDC, Atlanta).

Results

Variations in seropositivity

The age stratified sero-surveillance undertaken from 2011–13 among the children from seven different rural schools showed the in-apparent JE infection prevailing in the study area with infection rate ranging from 28.25–66.09%. A wide variation was observed in the infection rate among the subjects from different villages between JE and post-JE seasons. The JE seropositivity was significantly (p <0.05) higher in female than male during post-JE season (February 2012) (OR=1.65, 95% CI = 0.97–2.79). During other seasons (October 2011, 2012 and 2013 and February 2013) male and female positivity did not differ significantly (p >0.05). In 2012, seropositivity was significantly higher (p <0.001) in the JE season (October) than post-JE season (February) (OR = 5.15, 95% CI = 3.39–7.84). In 2013, positivity increased in post-JE season with respect to JE season of 2012 (OR = 1.138, 95% CI = 0.79–1.62), but it was not significant (p >0.05). In JE season of 2013, positivity decreased (but not significantly (p > 0.05) from post-JE season (OR = 0.853, 95% CI = 0.60–1.21). There was sudden increase in JE seropositivity during February to October 2012 and it was maintained thereafter. The seropositivity increased significantly (p <0.001) in JE season (October) during 2013 and 2012 in comparison to 2011. The seropositivity also increased significantly (p < 0.001) in the post-JE season of 2013 when compared to post-JE season in 2012.

Vector infection

A total of 7109 mosquitoes belonging to 14 species from six genera were collected. Among 14 species collected, Cx. tritaeniorhynchus dominated with per man hour density (PMHD) 176.79, constituting 89.53% of the total collection, followed by Cx. gelidus with PMHD 7.05 contributing 3.57%, while other culicines constituted 2.34%. Screening of mosquito samples from Thanjavur district showed one pool of Cx. tritaeniorhynchus positive, out of 25 pools and 1250 mosquitoes tested during December 2011; and one pool of Cx. tritaeniorhynchus positive, out of 51 pools and 2520 mosquitoes tested during July 2012.

Discussion

In India, serological surveys have provided invaluable data on the prevalence of antibodies for JE virus. A characteristic feature of JE is the occurrence of a large number of subclinical infections. Children in an endemic area are at serious risk of developing JE during the transmission season by the infective mosquito bites. The surveillance studies on JE conducted by CRME, Madurai, in Tamil Nadu in the past 20 yr have shown that vector abundance and vector MIR are good indicators for JE disease prevalence/occurrence[15]. Although, extensive paddy cultivation round the year in the Thanjavur district provide favourable breeding conditions for JE vectors, and being encircled by JE endemic neighbouring district, Cuddalore on northern and northwestern sides, only sporadic cases have been reported. A 2-year (1991 and 1992) prospective seroprevalence study[9] of comparable age-group in the district has reported low infection rates for JE, ranging from 2 to 5%. The main reason for this paradox was the uneven ratio between pigs and cattle, which was much higher (1: 400) when compared to the neighbouring endemic district, Cuddalore (1: 4) during that period[9]. This suggests that a lower pig and higher cattle density may be an important factor in limiting JE transmission in Thanjavur district. Hence, the higher population of JEV dampening (dead-end) host (Cattle) indirectly inhibits the process of spill over of the JE virus to humans. Although, there were no JE human case reports by the State Health Authorities till 2006, the silent JEV transmission since 2003 indicates that there is some shift or alteration taking place in the mode of JEV infection / activity in this non-endemic Thanjavur district.

Subsequently, a serosurvey conducted during the post-transmission season of February 2008 among school children (5–15 yr age group) in this district, showed that 27% population had JE infection, indicating an increase in the level of JEV infection rate (seroconversion rate ranged between 1.8 and 5.1%) compared to 1991–93 surveillance[16]. The infection rates observed here was lower than that observed in Cuddalore district (24–38%)[8]. This indicates that there is a steady increase in the build-up of infection rate even though no JE human cases are reported. The infection rates observed in this study were higher (32.2% for 2011–12 and 65.2% for 2012–13) than the previous 1991–93 studies, indicating a significant difference (p < 0.001) between them [Table 1].

The present JE seroprevalence study conducted in Thanjavur district, Tamil Nadu revealed that the school children are actually exposed to JE virus infection and have the grave risk of developing JE during the transmission season by the infective mosquito bite. Human is infected by the virus when the transmission cycle is complete. Species responsible for the transmission is Cx. tritaeniorhynchus which dominated the catch, and infections were detected from this species. Zoophagic effect of vectors with abundant cattle population divert Cx. tritaeniorhynchus mosquitoes from less preferred hosts like human and pig and are considered as an important factor which may reduce the risk of infection and spread of JE. Now-a-days, along with cattle rearing pig rearing has also become a source of income in these areas. Contrary to the earlier observations, the pigs: cattle ratio has narrowed down to 1 : 123 which might be the reason for increase in the JEV infection among the children population compared to 20 yr before.

Conclusion

The present study uncovered the actual situation prevailing among the young children in the study area, who are gravely at risk of developing JE during the transmission season resulting in high seroconversion rates, after the transmission season. Culex tritaeniorhynchus, a predominant species in this area plays an important role in the maintenance and transmission of JE virus and these mosquitoes are a concern for public health. Under such situation, the underlying contextual determining factors, mainly environmental aspects may cause spill over effects into the human population and trigger epidemics any time. This trend requires urgent attention which could be prevented with effective surveillance systems and vaccines. It is essential that appropriate control measures with intersectoral collaboration should be taken up based on sound knowledge of epidemiology and continuous surveillance strategies.

Acknowledgements

The authors are grateful to the Secretary, DHR and Director General, ICMR; and Director, CRME for providing the necessary support and facilities to undertake this study. Authors are greatly indebted to the medical and technical staff of the Department of Public Health and Preventive Medicine (DPH & PM), Tamil Nadu for their assistance during the field visits. Sincere thanks are due to all the supporting staff of CRME, Madurai, the school children, their parents and teachers, doctors and public health workers for their help and cooperation.

Statistical Handbook–2017: Agriculture—4.3: Area, production and productivity of principal crops in Tamil Nadu. Anna Salai, Chennai: Department of Economics and Statistics, Government of Tamil Nadu 2017. Available from:http://www.tn.gov.in/deptst/agriculture.pdf(Accessed on May 2, 2017).

Gajanana A, Thenmozhi V, Philip Samuel P, Reuben R. A community-based study of subclinical flavivirus infections in children in an area of Tamil Nadu, India, where Japanese encephalitis is endemic. Bull World Health Organ 1995; 73: 237–44.